A polyetheretherketone implant with time-regulated bone defect repair function, its preparation method and application

Abstract

This invention discloses a polyetheretherketone (PEEK) implant with time-dependent regulation of bone defect repair, its preparation method, and its applications, relating to the field of biomedical materials technology. The PEEK implant comprises: a substrate with a micro / nano topological structure on its surface, and a ROS-responsive multifunctional peptide chain loaded on the micro / nano topological structure; the substrate is made of PEEK or a PEEK composite material; the ROS-responsive multifunctional peptide chain comprises: a first functional short peptide with anti-inflammatory activity, a second functional short peptide with pro-angiogenic and / or bone differentiation-promoting effects, and a ROS-responsive molecule connecting the first and second functional short peptides; the second functional short peptide is connected to the micro / nano topological structure. This invention achieves multi-stage synergistic regulation of the entire bone regeneration process, significantly improving its in vivo bone integration efficiency and bone defect repair effect while maintaining the excellent mechanical properties of PEEK.

Description

Technical Field

[0001] This invention relates to the field of biomedical materials technology, and in particular to a polyetheretherketone implant with time-regulated bone defect repair function, its preparation method and application. Background Technology

[0002] Bone defect repair is one of the major challenges in orthopedic clinics, especially for critical-sized bone defects that cannot heal on their own and must rely on implants. Currently used metal implants (such as titanium alloys and cobalt-chromium alloys) have sufficient mechanical strength, but their elastic modulus is significantly higher than that of human bone, which can easily lead to a "stress shielding effect," causing surrounding bone resorption and implant loosening, thus affecting long-term stability.

[0003] Polyetheretherketone (PEEK), a high-performance polymer material, has an elastic modulus similar to that of human cortical bone, effectively reducing stress shielding. It also possesses excellent biocompatibility, chemical stability, and radiation permeability, and has been applied in implantation fields such as spinal, joint, and maxillofacial surgery. However, PEEK's surface is bioinert and lacks osteogenic activity. After implantation, it is easily encapsulated by fibrous tissue, making it difficult to form direct and strong osseointegration with bone tissue. This is a key issue limiting its further clinical application.

[0004] To improve the bioactivity of PEEK, existing technologies mainly focus on surface modification, such as introducing osteogenic components through plasma treatment, spraying hydroxyapatite coatings, or surface grafting of biomolecules. However, these methods often only achieve single-stage biological effects and cannot be temporally regulated according to the biological needs of different healing stages (such as early anti-inflammation, angiogenesis, and late osteogenic mineralization) during bone repair. Therefore, their effectiveness in promoting the integrated structural and functional regeneration of complex bone defects, especially critical-sized bone defects, is limited.

[0005] Therefore, existing technologies lack a method that can precisely regulate the surface bioactivity of PEEK in a temporal and multi-stage manner while maintaining its mechanical advantages, making it difficult to meet the dynamically changing physiological and healing needs during bone defect repair. Summary of the Invention

[0006] In view of the shortcomings of the prior art, the purpose of this invention is to provide a polyetheretherketone implant with time-regulated bone defect repair function, its preparation method and application, aiming to solve the problem of insufficient bone integration capacity of existing polyetheretherketone implants due to surface bioinertness.

[0007] The technical solution of the present invention is as follows: In a first aspect, a polyetheretherketone implant with time-regulated bone defect repair function is provided, comprising: a substrate with a micro-nano topological structure on its surface, and a ROS-responsive multifunctional peptide chain loaded on the micro-nano topological structure; The substrate is made of polyetheretherketone or a composite material of polyetheretherketone. The ROS-responsive multifunctional peptide chain includes: a first functional short peptide with anti-inflammatory activity, a second functional short peptide with pro-angiogenic and / or bone differentiation-promoting activity, and a ROS-responsive molecule connecting the first functional short peptide and the second functional short peptide; the second functional short peptide is connected to the micro / nano topological structure.

[0008] In a preferred embodiment, the amino acid sequence of the first functional short peptide is SESSE. The amino acid sequence of the second functional short peptide is REDV and / or YGFGG; The structure of the ROS-responsive molecule is as follows: ; The ROS-responsive molecule undergoes esterification and amide condensation reactions with the first and / or the second functional short peptides, respectively, via hydroxyl and carboxyl groups.

[0009] In a preferred embodiment, the ROS-responsive multifunctional peptide chain is selected from one or more of the following structures: .

[0010] In a preferred embodiment, the second functional short peptide and the micro / nano topological structure are connected by an intermediate bridging molecule. The structure of the intermediate bridging molecule is as follows: ; The intermediate bridging molecule undergoes a click chemistry reaction with the second functional short peptide via the DBCO group, and is covalently grafted with the micro / nano topology via the catechol group.

[0011] In a preferred embodiment, the micro / nano topological structure is selected from one or more of the following: micro / nano cone-shaped arrays, nanotube arrays, nanopore arrays, nanofiber mesh structures, micron-scale trenches, and crater-like pits.

[0012] In a preferred embodiment, the method for preparing the micro / nano topological structure is selected from one or more of oxygen plasma etching, ion beam etching, laser ablation, chemical etching, vapor deposition, and electrochemical deposition.

[0013] In a preferred embodiment, the morphology of the base is selected from one of the following: bone plate, screw, interbody fusion device, maxillofacial prosthesis, or oral implant.

[0014] In a preferred embodiment, the polyetheretherketone composite material is selected from one or more of carbon fiber reinforced polyetheretherketone and graphene modified polyetheretherketone.

[0015] In a second aspect, a method for preparing the polyetheretherketone implant described in the first aspect is provided, comprising the steps of: Provide a substrate with a surface having a micro / nano topological structure; ROS-responsive multifunctional peptide chains were loaded onto the micro / nano topology to obtain polyetheretherketone implants.

[0016] A preferred technical solution, wherein the method for preparing the substrate with a micro / nano topological structure on its surface includes the following steps: Polystyrene microsphere monolayers were prepared on the substrate surface using a microsphere inclined plane induced self-assembly method. Using the polystyrene microsphere monolayer as a sacrificial template, a substrate with a micro / nano cone-shaped array on the surface was prepared by oxygen plasma etching.

[0017] The application of polyetheretherketone implants as described in the first aspect in the preparation of bone defect repair materials.

[0018] The beneficial effects of the above technical solution of the present invention are as follows: (1) Achieving intelligent and time-sequential regulation of the entire bone healing process: This invention breaks through the limitations of traditional PEEK modification technology, which has a single function and static release. The implant can automatically switch its dominant biological functions on the surface according to the healing process in the body (inflammatory phase → repair phase → remodeling phase), from early anti-inflammatory, mid-term angiogenesis / osteoogenesis to late-term mineralization promotion, perfectly matching the physiological repair rhythm and greatly improving the repair efficiency and quality of critical-sized bone defects.

[0019] (2) Significantly improves the osseointegration performance of PEEK implants: Through the synergistic effect of chemical (ROS-responsive multifunctional peptide chains) and physical (micro-nano topological structures) signals, this invention not only promotes the initiation of early bone healing, but also ensures the direct and firm bonding between the new bone and the implant interface in the later stage, effectively solving the problem of fiber encapsulation caused by the biological inertness of PEEK and reducing the risk of aseptic loosening.

[0020] (3) While maintaining the inherent advantages of PEEK, it is endowed with bioactivity: all modifications are limited to the very thin surface layer, without changing the excellent mechanical properties (such as elastic modulus, strength), chemical stability and radiation permeability of the PEEK matrix, thus ensuring the long-term mechanical stability of the implant and the convenience of postoperative imaging assessment. Attached Figure Description

[0021] Figure 1This is a schematic diagram of the present invention; wherein, A is a schematic diagram of the ROS-responsive multifunctional peptide chain structure; B is a schematic diagram of the preparation process of the polyether ether ketone implant; and C is a schematic diagram of related verification.

[0022] Figure 2 This is a schematic diagram of the process for constructing a polyetheretherketone implant with time-regulated bone defect repair function according to the present invention.

[0023] Figure 3 This is a schematic diagram of the ROS-responsive multifunctional peptide chain structure and its ROS-responsive reaction of the present invention.

[0024] Figure 4 This is a schematic diagram illustrating the structure and function of the intermediate bridging molecule of the present invention.

[0025] Figure 5 This is a synthetic route diagram of the intermediate bridging molecule of the present invention.

[0026] Figure 6 These are schematic diagrams and results of the inclined plane-induced self-assembly of polystyrene microspheres according to the present invention; wherein, A is a schematic diagram of the inclined plane-induced self-assembly of microspheres; B is the transfer of microspheres to the PEEK surface by the impregnation-coating method.

[0027] Figure 7 This is a technical roadmap of the present invention.

[0028] Figure 8 These are images showing the results of the inclined plane-induced self-assembly of polystyrene microspheres according to the present invention; wherein, A is a photograph of the self-assembly of PS microspheres of different sizes on the water surface; B is a SEM image of the self-assembly of PS microspheres of different sizes on the PEEK surface.

[0029] Figure 9 These are SEM images of MC-PEEK samples of different sizes according to the present invention.

[0030] Figure 10 The images show the results of MC3T3-E1 cells on MC-PEEK samples of different sizes; where A is a SEM image of cell morphology; B is a statistical image of cell area; and C is a statistical image of cell viability on days 1, 3, and 5.

[0031] Figure 11 The images show the results of osteogenic induction of MC3T3-E1 cells on the surface of MC-PEEK samples of different sizes for 7 days; where A is the ALP staining result and B is the ALP quantitative detection result.

[0032] Figure 12 The images show the results of osteogenic induction of MC3T3-E1 cells on the surface of MC-PEEK samples of different sizes for 21 days; where A is the result of Alizarin Red staining and B is the result of semi-quantitative detection of calcium deposition.

[0033] Figure 13 This is a graph showing the gene expression results of MC3T3-E1 cells after osteogenic induction on MC-PEEK samples of different sizes for 7 and 14 days.

[0034] Figure 14 This is a diagram showing the regulatory effect of the PEEK temporal function table / interface on RAW264.7 macrophages; where A is the SEM morphology of cells on the sample surface; B is the immunofluorescence staining result of CD163 antibody (M2 type); C is the result of flow cytometry detection of the number of CD86 (M1 type) and CD206 (M2 type) positive cells; and D is the result of RT-qRCR detection of the expression of related inflammatory factors.

[0035] Figure 15 This is a diagram showing the osteogenic differentiation results of BMSC cells at the PEEK time-series functional table / interface; where A is the ALP staining result on day 14; B is the ALP quantitative detection result; C is the Alizarin Red staining result on day 21; D is the semi-quantitative detection result of calcium deposition; and E is the result of RT-qRCR detection of osteogenic differentiation-related gene expression. Detailed Implementation

[0036] This invention provides a polyetheretherketone (PEEK) implant with time-regulated bone defect repair function, its preparation method, and its application. The core of this invention lies in endowing the bio-inert PEEK surface with dynamic intelligent response capabilities, enabling it to actively participate in and guide the entire bone healing process. To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention is further described in detail below.

[0037] This invention provides a polyetheretherketone implant with time-regulated bone defect repair function, comprising: a substrate with a micro-nano topological structure on its surface, and a ROS-responsive multifunctional peptide chain loaded on the micro-nano topological structure; Wherein, the substrate is polyetheretherketone or a polyetheretherketone composite material; The ROS-responsive multifunctional peptide chain includes: a first functional short peptide with anti-inflammatory activity, a second functional short peptide with pro-angiogenic and / or bone differentiation-promoting activity, and a ROS-responsive molecule connecting the first functional short peptide and the second functional short peptide; the second functional short peptide is connected to the micro / nano topological structure.

[0038] Specifically, this invention addresses the problem of insufficient bone integration capacity in polyetheretherketone (PEEK) implants due to their surface bioinertness, and provides a PEEK implant capable of temporally regulating the bone defect repair process. The basic idea of ​​this invention is to construct a smart interface on the surface of the PEEK implant through micro / nano topological structure construction and active peptide molecule modification, enabling it to respond to the biological microenvironment at different stages of bone repair and sequentially exert functions of "anti-inflammatory / reactive oxygen species scavenging - pro-angiogenesis / osteoogenesis - pro-mineralization".

[0039] This approach enables the implant to respond to the inflammatory microenvironment through a first functional short peptide in the early stages of repair, inhibiting excessive inflammation and scavenging reactive oxygen species; in the middle stages of repair, it simultaneously promotes angiogenesis and osteogenic differentiation through a second functional short peptide; and in the later stages of repair, it further guides bone matrix deposition and mineralization through surface micro- and nano-topological structures. This invention achieves multi-stage synergistic regulation of the entire bone regeneration process, significantly improving its in vivo bone integration efficiency and bone defect repair effect while maintaining the excellent mechanical properties of PEEK.

[0040] In some embodiments, the amino acid sequence of the first functional short peptide is SESSE, but it is not limited to this. Other peptides with clear anti-inflammatory effects can be used instead, such as short peptides derived from Ac2-26 (Annexin A1 mimic peptide).

[0041] In some embodiments, the amino acid sequence of the second functional short peptide is REDV and / or YGFGG, but is not limited thereto. In addition to VEGF mimic peptides, stromal cell-derived factor-1α (SDF-1α) mimic peptides can be used to promote angiogenesis. In addition to BMP-2 mimic peptides, osteogenic peptides can be fragments of parathyroid hormone-related protein (PTHrP), adhesion peptides derived from osteocoagulin (BSP) or osteopontin (OPN).

[0042] In addition, multiple peptide chains with different single functions can be grafted, such as one RGD adhesion peptide and another BMP-2 mimic peptide, which can share the same response unit or have their own independent response / release mechanisms.

[0043] In some embodiments, the structure of the ROS-responsive molecule is as follows: However, this is not the only option; other chemical bonds that can break under physiologically relevant concentrations of ROS (especially H2O2) can be used as substitutes, such as pinacol borate, selenium bonds, tellurium bonds, and peroxyoxalate. The response threshold can be adjusted through chemical modification to match ROS levels with different degrees of deficiency. The ROS-responsive molecule can form a ROS-responsive multifunctional peptide chain by esterification and amide condensation reactions with the first and / or second functional short peptides, respectively, through hydroxyl and carboxyl groups. This ROS-responsive multifunctional peptide chain can break chemical bonds in the presence of ROS. Figure 3 ).

[0044] In some embodiments, the ROS-responsive multifunctional peptide chain is selected from one or more of the following structures: .

[0045] In some embodiments, the second functional short peptide and the micro / nano topology are connected via an intermediate bridging molecule; The structure of the intermediate bridging molecule is as follows: ; The intermediate bridging molecule undergoes a click chemical reaction with the second functional short peptide through the DBCO group, and is covalently grafted with the micro / nano topological structure through the catechol group, but is not limited thereto. Specifically, alternative methods for surface grafting chemistry include: (1) Other click chemistry: copper-free catalytic systems such as tetrazine-trans-cyclooctene (TCO) click chemistry, thiol-ene click chemistry, and oxadionene-tetrazole click chemistry can be used, which have better biocompatibility. (2) Traditional coupling chemistry: after introducing appropriate functional groups, the peptide chain can be fixed by carbodiimide (EDC / NHS) condensation, glutaraldehyde crosslinking, maleimide-thiol reaction, etc. (3) Biomimetic anchoring: using the self-polymerization and adhesion properties of dopamine or its derivatives on the PEEK surface, a polydopamine coating is formed. This coating is rich in active groups and can be further grafted with peptide chains through Michael addition or Schiff base reaction.

[0046] In some embodiments, the micro / nano topology is selected from one or more of micro / nano cone-shaped arrays, nanotube arrays, nanopore arrays, nanofiber mesh structures, micron-scale trenches, and crater-like pits.

[0047] In some embodiments, the preparation method of the micro / nano topological structure is selected from one or more of oxygen plasma etching, ion beam etching, laser ablation, chemical etching, vapor deposition, and electrochemical deposition, but is not limited thereto. Specifically, for example, similar or different micro / nano topological structures (such as nanopillars, nanopores, and nanowrinkles) can be constructed on the substrate surface using methods such as oxygen plasma etching, ultraviolet / ozone treatment, ion beam etching, and laser ablation; or a porous network structure can be formed on the substrate surface by sulfonation with concentrated sulfuric acid followed by hydrothermal treatment, which also has a high specific surface area and osteogenic capacity; or 3) a basic roughness is first constructed using the above methods, and then nanoparticles (such as zinc oxide and titanium dioxide) are grown on it by vapor deposition, electrochemical deposition, etc., to form a composite micro / nano topological structure.

[0048] In some embodiments, the morphology of the base is selected from one of a bone plate, screw, interbody fusion device, maxillofacial prosthesis, or oral implant.

[0049] In some embodiments, the polyetheretherketone composite material is selected from one or more of carbon fiber reinforced polyetheretherketone and graphene modified polyetheretherketone, but is not limited thereto. Other materials whose surfaces can be activated and grafted can also be used, and can be selected according to actual needs.

[0050] In one specific embodiment, the polyetheretherketone implant of the present invention with time-regulated bone defect repair function includes: a substrate having a micro / nano cone-shaped array structure on its surface, and a ROS-responsive multifunctional peptide chain loaded on the micro / nano cone-shaped array structure. The substrate is made of polyetheretherketone (PEEK). The ROS-responsive multifunctional peptide chain is selected from one or more of the following structures: ; The ROS-responsive multifunctional peptide chain and the micro / nano cone-shaped array structure are connected by an intermediate bridging molecule. The structure of the intermediate bridging molecule is as follows: ; The intermediate bridging molecule undergoes a click chemistry reaction with the second functional short peptide via the DBCO group, and is covalently grafted with the micro / nano topology via the catechol group.

[0051] Specifically, this solution provides a PEEK implant with time-regulated bone defect repair function. The implant consists of a PEEK substrate and functionalized interfaces constructed on its surface. The fabrication process is as follows: Figure 1 As shown. The functional interface includes: (1) Micro-nano cone-shaped array structure: directly constructed on the surface of PEEK substrate. The structure is first constructed by self-assembly of monolayer microspheres on the surface of PEEK substrate to build a "sacrificial template", and then formed by oxygen plasma etching. Its size is in the nanometer to micrometer range, and it is sharp cone-shaped with high specific surface area and roughness.

[0052] (2) Covalently grafted ROS-responsive multifunctional peptide chains: These are firmly grafted onto the surface of the aforementioned micro / nano structures via bioorthogonal click chemistry. The ROS-responsive multifunctional peptide chain layer involves two linear molecules, each with a core structure comprising three parts, such as... Figure 1 As shown in A: (a) First functional short peptide (front end): has anti-inflammatory function. For example, a specific oligopeptide sequence that can induce macrophages to polarize to the M2 type (anti-inflammatory / repair type) can be selected (such as the ultrashort peptide SESSE sequence with anti-inflammatory activity).

[0053] (b) ROS-responsive linker: a chemical bond or molecular fragment that connects the first and second functional peptides and can be broken in the presence of ROS (such as H2O2, •OH). For example, the bridging molecule HPBA-DOPAC (4-hydroxymethylphenylboronic acid-3,4-dihydroxyphenylacetic acid) with arylboronic acid ester bonds in this scheme.

[0054] (c) Secondary functional short peptides (rear end): These have the function of promoting angiogenesis or promoting bone differentiation. For example, derivative short peptides that promote endothelial cell adhesion and proliferation—REDV sequence and active fragments of osteogenic growth peptide (OGP)—YGFGG sequence can be selected.

[0055] (3) Design of intermediate bridging molecule: Design an intermediate bridging molecule (DOPA)4-PEG3-DBCO, which is composed of 4 catechol and dibenzocyclooctylene (DBCO) groups (chemical structure as shown in Figure 1). Figure 4 (As shown). The catechol structure on one side of the intermediate bridging molecule can be modified onto a substrate with a micro / nano cone-shaped array structure via non-covalent bonding; the DBCO group on the other side can undergo an azido-acetylene cycloaddition (SPAAC) reaction with the two azido-modified active peptide chains via click chemistry, fixing the peptide chains onto the surface of the aforementioned micro / nano cone-shaped array structure. Furthermore, an HPBA-DOPAC small molecule structure containing a borate ester bond was designed in the middle of the two peptide chains, which can respond to ROS and release the anti-inflammatory short peptide sequence SESSE.

[0056] The working principle of this invention is as follows: In the early stage of implantation, the high concentration of ROS at the defect site triggers the breakage of responsive linker units, leading to the rapid release of the first functional short peptide, which exerts local anti-inflammatory and ROS scavenging effects, improving the healing microenvironment. Subsequently, the exposed or slowly released second functional short peptide continues to act on the surrounding tissues, promoting the migration, proliferation, and differentiation of vascular endothelial cells and osteoblastic progenitor cells. Finally, the unreleased peptide chain residues and the underlying micro / nano-cone structures provide long-term, stable biophysical signals to the cells, guiding extracellular matrix deposition and heterogeneous nucleation of hydroxyapatite crystals, achieving robust osteointegration.

[0057] The specific effects of each component are as follows: (1) Effects of the micro / nano cone-shaped array structure: (a) Increased specific surface area and loading capacity: provided huge binding sites for subsequent peptide chain modification, significantly increased the surface loading density of functional molecules, and enhanced biological effects.

[0058] (b) Provides topological signals for cell orientation: The sharp, cone-shaped structures can mechanically stimulate cytoskeleton rearrangement, promote osteoblast adhesion, spread and differentiation, and inhibit excessive proliferation of fibroblasts, thereby physically inhibiting fibrous capsule formation.

[0059] (c) Promotes biomineralization: Micro- and nano-scale roughness and specific crystal nuclei provide ideal nucleation sites for the deposition of hydroxyapatite, accelerating the mineralization process at the bone interface.

[0060] (2) Effects of ROS-responsive peptide chain design: (a) Precise “on-demand release”: Utilizing the characteristic of ROS overexpression in the early inflammatory microenvironment as a biological trigger, the timing of the release of anti-inflammatory function is highly consistent with the pathological needs, thus achieving precision and efficiency in treatment.

[0061] (b) Automatic and orderly switching of functions: The transition from front-end functions to back-end functions is achieved naturally and reliably through the breaking of a response bond on a peptide chain, without the need for external intervention. The system is simple and reliable.

[0062] (c) Multifunctional integration and synergy: Functions such as anti-inflammatory, angiogenesis and osteoproliferation that originally required multiple administrations or complex carriers to achieve are integrated into a single molecular system, achieving synergistic effects in time and space.

[0063] (3) The effect of the bioorthogonal click chemical modification strategy: (a) Highly efficient and specific grafting: The reaction conditions are mild, fast, and yield high. It can be carried out selectively in the presence of complex biomolecules, ensuring that the peptide chain is firmly fixed on the surface in the preset conformation and density, and its function is not easily lost.

[0064] (b) Good controllability and universality: This method facilitates quantitative control of surface grafting density, and the strategy can be widely applied to the surface of other implant materials that require the fixation of biomolecules.

[0065] Based on the same inventive concept, embodiments of the present invention provide a method for preparing a polyetheretherketone implant as described above, comprising the following steps: Provide a substrate with a surface having a micro / nano topological structure; ROS-responsive multifunctional peptide chains were loaded onto the micro / nano topology to obtain polyetheretherketone implants.

[0066] In some embodiments, the method for preparing the substrate with a micro / nano topological structure on its surface includes the steps of: Polystyrene microsphere monolayers were prepared on the substrate surface using a microsphere inclined plane induced self-assembly method. Using the polystyrene microsphere monolayer as a sacrificial template, a substrate with a micro / nano cone-shaped array on the surface was prepared by oxygen plasma etching.

[0067] In one specific implementation, such as Figure 2 As shown, the method for preparing the polyetheretherketone implant of the present invention includes the following steps: (1) PEEK substrate pretreatment: Polish the PEEK substrate to a near mirror finish on one side, and then ultrasonically clean it with organic solvents (such as acetone and ethanol) to remove surface contaminants. Dry it for later use.

[0068] (2) Construction of micro / nano cone-shaped array structure: (a) Polystyrene (PS) microsphere monolayers were prepared on a PEEK surface using a microsphere inclined plane induced self-assembly method. For example... Figure 6 As shown, a tilted glass slide was placed on a culture dish filled with pure water. A 2wt% 2μm polystyrene (PS) microsphere suspension (water:ethanol = 1:1 (v / v)) was dropped onto the glass slide. The PS microspheres spread on the water surface and automatically formed a monolayer.

[0069] (b) The PS microspheres assembled on the water surface were transferred to the surface of the PEEK substrate by the "immersion-pulling" method to prepare the "sacrificial template" required for plasma etching.

[0070] (c) The pretreated PEEK substrate with the “sacrificial template” is placed in the reaction chamber of the oxygen plasma treatment device.

[0071] (d) Etching under specific process parameters: control oxygen flow rate (10-100 sccm), chamber pressure (10-100 Pa), radio frequency power (50-300 W) and processing time (1-30 minutes).

[0072] (e) By anisotropically bombarding and chemically etching the PEEK surface with high-energy active particles in plasma, a uniform and high-density cone-like microarray (MC) is formed in situ on the surface, resulting in a substrate with a micro-nano topological structure, denoted as MC-PEEK sample.

[0073] (3) Surface activation and introduction of click chemical handles: The synthetic route of the intermediate bridging molecule (DOPA) 4-PEG3-DBCO is as follows: Figure 5 As shown. The above MC-PEEK sample was treated with oxygen plasma and then immersed overnight in a phosphate buffer solution (pH 8.5) of 0.1 mg / mL (DOPA)4-PEG3-DBCO to obtain the DBCO-MC-PEEK sample. The intermediate bridging molecule was modified on the surface of the MC-PEEK sample through the catechol group in the structure, providing a reaction site for subsequent grafting of multifunctional peptide chains.

[0074] (4) Modification of ROS-responsive multifunctional peptide chains: (a) To enable click chemistry with the DBCO group in the aforementioned intermediate bridging molecule, two ROS-responsive multifunctional peptide chains, N3-YGFGG-HPBA-DOPAC-SESSE and N3-REDV-HPBA-DOPAC-SESSE, with N-terminal azide groups modified, were custom-synthesized. The short peptide sequences SESSE, YGFGG, and REDV possess anti-inflammatory, osteogenic, and angiogenic effects, respectively. The intermediate link is composed of small molecules HPBA (4-hydroxyphenylboronic acid) and DOPAC (3,4-dihydroxyphenylacetic acid), which can specifically respond to ROS and undergo degradation. The degraded substrates HBA (p-hydroxybenzyl alcohol) and DOPAC (3,4-dihydroxyphenylacetic acid ester) have antioxidant effects, capable of scavenging excess ROS and thus alleviating inflammatory responses.

[0075] (b) The DBCO-MC-PEEK sample was placed in a mixed solution of the two ROS-responsive multifunctional peptides at 1 mg / mL and incubated at room temperature for 3 hours. The DBCO group can efficiently graft the multifunctional peptides onto the surface of the MC-PEEK sample via a specific SPAAC reaction with the azide group.

[0076] (c) After the reaction is complete, thoroughly wash to remove the physically adsorbed ROS-responsive multifunctional peptide chains and obtain the final functionalized PEEK implant, denoted as Peptide-MC-PEEK sample.

[0077] The ROS-responsive functional peptide chain is an independent functional molecular unit with the general structural formula ALB.

[0078] A: The first functional short peptide, its function is anti-inflammatory and regulating macrophage polarization towards the M2 phenotype.

[0079] L: ROS-responsive molecules, whose function is to specifically respond to ROS and degrade it. The degraded substrate also has antioxidant effects, which can remove excess ROS and thus alleviate the inflammatory response.

[0080] B: A short peptide with secondary functions, which promotes angiogenesis and bone differentiation.

[0081] In addition, the N-terminus of the peptide chain is modified with an azide group that can undergo click chemistry covalent bonding with DBCO.

[0082] Based on the same inventive concept, embodiments of the present invention provide the application of the polyetheretherketone implant described above in the preparation of bone defect repair materials.

[0083] The present invention will be further described below through specific embodiments.

[0084] Example 1 This invention provides a polyetheretherketone implant with time-regulated bone defect repair function, and tests its performance. The research technical route is as follows: Figure 7 As shown.

[0085] The specific preparation method of polyetheretherketone implants with time-regulated bone defect repair function is as follows: I. Self-assembly induced by inclined plane of polystyrene microspheres The PEEK substrate was polished to near mirror finish on one side, then ultrasonically cleaned sequentially with organic solvents (such as acetone and ethanol) to remove surface contaminants, and dried for later use. Polystyrene (PS) microsphere monolayers were then prepared on the PEEK surface using a microsphere inclined plane-induced self-assembly method. (Details follow...) Figure 6 As shown, a tilted glass slide was placed on a petri dish filled with pure water. A 2wt% 2μm polystyrene (PS) microsphere suspension (water:ethanol = 1:1 (v / v)) was dropwise added onto the slide, and the PS microspheres automatically spread on the water surface to form a monolayer. The PS microspheres assembled on the water surface monolayer were transferred to the surface of a PEEK substrate using the "immersion-coating" method to obtain the "sacrificial template" required for plasma etching. The SEM morphology of the PS microspheres on the PEEK surface is shown in the figure. Figure 8 As shown, PS microspheres of different sizes can self-assemble on the PEEK surface to form a single-layer dense topology.

[0086] II. Construction of Conical Spike-like Micro / Nano Array Structures of Different Sizes on PEEK Surface Further oxygen plasma etching was performed on the PEEK surface assembled from PS microspheres. By adjusting etching parameters, such as keeping the oxygen flow rate (30 sccm), chamber pressure (50 Pa), and RF power (150 W) constant, etching was performed for 15, 30, 45, and 60 minutes respectively for PS microsphere sizes ranging from 500 nm to 2000 nm, resulting in four different sized cone-shaped (MC) micro / nano array structures of PEEK surface, denoted as MC-PEEK. The SEM morphology of different samples is shown below. Figure 9 As shown, the surfaces of each group of PEEK samples exhibit a regularly arranged cone-shaped micro-nano array structure, and the spacing between the arrays increases with the increase of size.

[0087] III. Surface Activation and Introduction of Click Chemical Handle The synthetic route of the intermediate bridging molecule (DOPA) 4-PEG3-DBCO is as follows: Figure 5 As shown, the sample was custom-made from a chemical synthesis company. The MC-PEEK sample was treated with oxygen plasma and then immersed overnight in a phosphate buffer solution (pH 8.5) of 0.1 mg / mL (DOPA)4-PEG3-DBCO to obtain the DBCO-MC-PEEK sample. An intermediate bridging molecule was modified onto the surface of the MC-PEEK sample via the catechol group in the structure, providing a reaction site for subsequent grafting of multifunctional peptide chains.

[0088] IV. Modification of ROS-responsive multifunctional peptide chains (1) In order to enable click chemistry with the DBCO group in the above intermediate bridging molecule, two ROS-responsive multifunctional peptide chains N3-YGFGG-HPBA-DOPAC-SESSE and N3-REDV-HPBA-DOPAC-SESSE, respectively, modified with azide groups at the N-terminus, were synthesized. The short peptide sequences of SESSE, YGFGG, and REDV have anti-inflammatory, osteogenic, and angiogenic effects, respectively. The intermediate link is composed of small molecules HPBA (4-hydroxyphenylboronic acid) and DOPAC (3,4-dihydroxyphenylacetic acid), which can specifically respond to ROS and degrade. The degraded substrates HBA (p-hydroxybenzyl alcohol) and DOPAC (3,4-dihydroxyphenylacetic acid ester) have antioxidant effects and can scavenge excess ROS to alleviate the inflammatory response.

[0089] The specific structure of the ROS-responsive multifunctional peptide chain N3-YGFGG-HPBA-DOPAC-SESSE is as follows: .

[0090] The specific structure of the ROS-responsive multifunctional peptide chain N3-REDV-HPBA-DOPAC-SESSE is as follows: .

[0091] (2) The DBCO-MC-PEEK sample was placed in a mixed solution of the two ROS-responsive multifunctional peptide chains at 1 mg / mL and incubated at room temperature for 3 hours. The DBCO group can efficiently graft the multifunctional peptide chain onto the surface of the MC-PEEK sample through a specific SPAAC reaction with the azide group.

[0092] (3) After the reaction is complete, thoroughly wash to remove the physically adsorbed ROS-responsive multifunctional peptide chains and obtain the final functionalized PEEK implant, which is denoted as Peptide-MC-PEEK sample.

[0093] The following tests were conducted to determine the factors influencing the performance of the Peptide-MC-PEEK sample.

[0094] I. Effects of different sized cone-shaped micro / nano array structures on the morphology and viability of preosteoblasts MC3T3-E1 cells were seeded onto the surfaces of the four groups of MC-PEEK samples of different sizes. Figure 10 As shown in Figure A, cell spreading on the surface of MC-PEEK samples of different sizes was significantly inhibited, and this inhibition was size-dependent, showing that the cell spreading area gradually decreased with increasing size. Figure 10 (B) CCK-8 results are as follows Figure 10 As shown in Figure C, the cells maintained high proliferation activity on the surface of each group of samples, and the difference in cell proliferation rate between MC-PEEK samples of different sizes was negligible.

[0095] II. Effects of Conical-Spiked Micro / Nano Array Structures of Different Sizes on Preosteoblast Differentiation To screen for the optimal cone-shaped structure size for promoting osteogenic differentiation, MC3T3-E1 cells were seeded onto the surface of MC-PEEK samples of different sizes from the four groups mentioned above. On day 7 of osteogenic induction, ALP qualitative staining and quantitative determination were performed on the cells to evaluate the early and mid-stage osteogenic differentiation effects. The results are as follows: Figure 11As shown in Figure A, except for the 500nm sample group, the surfaces of all other MC-PEEK sample groups exhibited a darker color than the Flat group, and the ALP staining gradually deepened with increasing size. The quantitative results for ALP also showed a high degree of consistency with the staining results. Figure 11 (B)

[0096] On day 21 of osteogenic induction, alizarin red reagent was used to stain cells for calcium deposition to evaluate the late osteogenic differentiation effect. Results are as follows: Figure 12 As shown in Figure A, the surfaces of all MC-PEEK samples exhibited a darker color than those in the Flat group, and the calcium deposition staining gradually deepened with increasing size. Furthermore, the semi-quantitative results of calcium deposition also showed an increasing trend with increasing size. Figure 12 (B)

[0097] The expression levels of osteogenic differentiation-related genes (Runx2, ALP, Col 1α1, and OCN) in MC3T3-E1 cells were further detected using RT-qPCR. The results are as follows: Figure 13 As shown, on day 7 of osteogenic induction, the expression of genes related to early and mid-stage osteogenic differentiation (Runx2, ALP, Col 1α1) all exhibited a significant morphology-size dependence, meaning that the larger the size, the more pronounced the increase in gene expression levels. On day 14 of osteogenic induction, the expression levels of Runx2 and ALP genes decreased in all groups, while the expression levels of Col 1α1 and OCN genes significantly increased, and the expression of all four genes remained size-dependent.

[0098] Based on the above experimental results, a Peptide-MC-PEEK sample was prepared using MC-PEEK samples made from 2000nm PS microspheres, and the following experiments were conducted.

[0099] III. Regulation of macrophages by the PEEK temporal functional table / interface First, the morphological characteristics of RAW264.7 macrophages on different sample surfaces are as follows: Figure 14 As shown in Figure A, the cells in the PEEK group are round, while the cells in the MC-PEEK group and the Peptide-MC-PEEK group exhibit radial "tentacles," and the cell spreading area is relatively larger. The results of immunofluorescence staining ( Figure 14 (B) indicated that the Peptide-MC-PEEK group could upregulate the expression of CD163, a macrophage M2 marker; further flow cytometry analysis results ( Figure 14 The results of RT-qCR also showed that the Peptide-MC-PEEK group reduced the number of CD86-positive cells (M1 type) and increased the number of CD206-positive cells (M2 type). Figure 14As shown in Figure D, the Peptide-MC-PEEK group significantly downregulated the gene expression of pro-inflammatory cytokines IL-1β and TNF-α, and upregulated the gene expression of the anti-inflammatory cytokine IL-10. These results demonstrate that the PEEK temporal functional table / interface can play a role in regulating macrophage polarization and anti-inflammation.

[0100] IV. Regulation of osteogenic differentiation of bone marrow stem cells by the PEEK temporal functional table / interface Using mouse-derived BMSCs as the research subject, the qualitative staining results of ALP on day 14 of osteogenic induction were as follows: Figure 15 As shown in Figure A, the surface of the Peptide-MC-PEEK sample exhibits a distinct deep blue color, and the quantitative results of ALP are highly consistent with the staining results. Figure 15 (Middle B). Similarly, on day 21 of osteogenic induction, the alizarin red staining results were as follows: Figure 15 As shown in C, the surface of the Peptide-MC-PEEK sample exhibits a darker color than the other two groups, and the semi-quantitative results of calcium deposition are consistent with this. Figure 15 (D).

[0101] On days 7 and 14 of osteogenic induction, the expression levels of osteogenic differentiation-related genes (Runx2, ALP, Col 1α1, and OCN) in BMSCs were further detected by RT-qPCR. The results are as follows: Figure 15 As shown in Figure E, compared to the other two groups, the Peptide-MC-PEEK group significantly upregulated the expression of osteogenic differentiation-related genes Runx2, ALP, Col 1α1, and OCN. These results demonstrate that the PEEK temporal functional table / interface can play a role in regulating stem cell osteogenic differentiation.

[0102] In summary, the present invention provides a polyetheretherketone implant with time-regulated bone defect repair function, the core of which lies in its functionalized surface, which is a combination of "micro-nano topological structure" and "ROS-responsive multifunctional peptide chain", and this combination is designed to realize the aforementioned three-stage time-regulated repair function.

[0103] Compared with known existing technologies (such as plasma treatment, HA coating, single biomolecule grafting, etc.), the key difference of this invention is that it elevates the surface modification of PEEK implants from a static, single-function level to an "intelligent" biointerface system that can dynamically respond to and actively regulate different stages of bone healing.

[0104] Specifically, this can be broken down into the following three interrelated and synergistic key technological innovations: (1) Design concept of temporal functional regulation: Unlike existing technologies that only focus on the ultimate goal of "promoting bone formation", this invention proposes and constructs a phased and progressive functional regulation strategy for the entire process of bone healing (inflammatory phase, repair / regeneration phase, and remodeling phase) of "anti-inflammatory / ROS clearance → angiogenesis / osteoogenesis → guided mineralization". This is a fundamental innovation in design thinking.

[0105] (2) Construction and grafting of ROS-responsive multifunctional peptide chains: (a) Response mechanism: Its release behavior is triggered by excessive reactive oxygen species (ROS) in the early defect microenvironment, realizing the "on-demand matching" of functional release and physiological needs.

[0106] (b) Functional arrangement: A single peptide chain integrates multifunctional sequences with different front and back ends, which are linked together by responsive linkages to achieve precise timing control of the release of the front end function (anti-inflammatory) first and the back end function (pro-angiogenic / osteogenic) later.

[0107] (c) Fixation strategy: Use bioorthogonal click chemistry for surface grafting to ensure that the modification process is efficient, specific and has little impact on the activity of biomolecules.

[0108] (3) Synergistic coupling of micro / nano structures and biochemical modifications: Instead of simply modifying active molecules on a flat surface, micro / nano topological structures are first constructed as a physical substrate. This structure not only provides physical guidance for subsequent cell adhesion and mineralization (later function), but more importantly, its high specific surface area enhances the loading of active molecules and provides unique topological stimulation to cells, producing a synergistic enhancement effect with biochemical signals.

[0109] It should be understood that the application of the present invention is not limited to the examples above. Those skilled in the art can make improvements or modifications based on the above description, and all such improvements and modifications should fall within the protection scope of the appended claims.

Claims

1. A polyether ether ketone implant with a time-dependent regulation of bone defect repair function, characterized in that, include: A substrate with a micro / nano topological structure on its surface, and a ROS-responsive multifunctional peptide chain loaded on the micro / nano topological structure; The substrate is made of polyetheretherketone or a composite material of polyetheretherketone. The ROS-responsive multifunctional peptide chain includes: a first functional short peptide with anti-inflammatory activity, a second functional short peptide with pro-angiogenic and / or bone differentiation-promoting activity, and a ROS-responsive molecule connecting the first functional short peptide and the second functional short peptide; the second functional short peptide is connected to the micro / nano topological structure.

2. The polyether ether ketone implant according to claim 1, characterized in that The amino acid sequence of the first functional short peptide is SESSE; The amino acid sequence of the second functional short peptide is REDV and / or YGFGG; The structure of the ROS-responsive molecule is ; The ROS-responsive molecule undergoes esterification and amide condensation reactions with the first and / or the second functional short peptides, respectively, via hydroxyl and carboxyl groups.

3. The polyether ether ketone implant of claim 1, wherein, The ROS-responsive multifunctional peptide chain is selected from one or more of the following structures: 。 4. The polyether ether ketone implant according to claim 3, characterized in that The second functional short peptide and the micro / nano topology are connected by an intermediate bridging molecule; The structure of the intermediate bridging molecule is ; The intermediate bridging molecule undergoes a click chemistry reaction with the second functional short peptide via the DBCO group, and is covalently grafted with the micro / nano topology via the catechol group.

5. The polyether ether ketone implant of claim 1, wherein, The micro / nano topological structure is selected from one or more of the following: micro / nano cone-shaped arrays, nanotube arrays, nanopore arrays, nanofiber mesh structures, micron-scale trenches, and crater-like pits.

6. The polyether ether ketone implant of claim 1, wherein, The method for preparing the micro-nano topological structure is selected from one or more of oxygen plasma etching, ion beam etching, laser ablation, chemical etching, vapor deposition, and electrochemical deposition.

7. The polyether ether ketone implant of claim 1, wherein, The shape of the base is selected from one of the following: bone plate, screw, interbody fusion device, maxillofacial prosthesis or oral implant; The polyetheretherketone composite material is selected from one or more of carbon fiber reinforced polyetheretherketone and graphene modified polyetheretherketone.

8. A method of producing a polyether ether ketone implant according to claim 1, characterized in that Including the following steps: Provide a substrate with a surface having a micro / nano topological structure; ROS-responsive multifunctional peptide chains were loaded onto the micro / nano topology to obtain polyetheretherketone implants.

9. The method for preparing the polyetheretherketone implant according to claim 8, characterized in that, The method for preparing the substrate with a micro / nano topological structure on its surface includes the following steps: Polystyrene microsphere monolayers were prepared on the substrate surface using a microsphere inclined plane induced self-assembly method. Using the polystyrene microsphere monolayer as a sacrificial template, a substrate with a micro / nano cone-shaped array on the surface was prepared by oxygen plasma etching.

10. The use of the polyetheretherketone implant as described in any one of claims 1-7 in the preparation of bone defect repair materials.

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